The platelet membrane glycoprotein Ib-IX complex (GPIb-IX) plays an important role in platelet adhesion, particularly in arteries and capillaries. At sites of vascular injury, GPIb-IX interacts with the subendothelium-bound von Willebrand factor (VWF). This interaction not only mediates the physical adherence of platelets to the site of vascular injury, but also initiates signaling. GPIb-IX-mediated signaling leads to the cytoskeleton reorganization and activation of the ligand binding function of the platelet integrin aIIbB3 that mediates stable platelet adhesion, spreading and aggregation. This process is critical in both hemostasis and thrombosis. VWF binding to GPIb-IX also induces release of platelet microparticles that facilitates coagulation. Under certain pathological conditions such as thrombotic thrombocytopenic purpura (TTP), binding of GPIb-IX to circulating ultra-large VWF multimers induces microthrombosis and development of microthrombopathy. The overall goals of this project are to understand the signaling mechanism of GPIb-IX in thrombosis, platelet activation and cytoskeleton organization, and to explore the possibility of developing GPIb-IX-based therapeutic approaches. In the last 3 years' since the previous renewal, we have made significant progresses in understanding the regulatory role of 14-3-3 in GPIb function, developed an in vivo inhibitor delivery method allowing testing the in vivo effects of 14-3-3 inhibitor on GPIb function and thrombus formation, and delineated a novel Lyn-PI3K- Akt1/Akt2-NO-cGMP signaling pathway as an early signaling mechanism leading to integrin activation and stable platelet adhesion under shear stress. Furthermore, we have obtained substantial amount of preliminary data that forms the basis of new hypotheses for the specific aims of this competitive renewal. We propose following specific aims: Aim 1, to determine the role of GPIb-IX and 14-3-3 in thrombosis and microvascular thrombosis in sepsis and TTP and to explore the therapeutic effect of an inhibitor of GPIb-IX-14-3-3 interaction; Aim 2, to investigate the role of LIMK1 in GPIb-IX-mediated platelet activation and cytoskeleton reorganization; and Aim 3, to determine the role of Rac1 in GPIb-IX-mediated signaling leading to platelet activation and microparticle release.